BACKGROUND: Gilbert's syndrome (GS) is a hereditary disorder caused by mutations in the UGT1A1 gene, leading to unconjugated hyperbilirubinemia. Accurate detection of UGT1A1 gene polymorphisms is crucial for diagnosis and management of GS. This study evaluated the clinical application of pyrophosphorolysis-activated polymerization PCR (PAP-PCR) method in dection of UGT1A1 gene polymorphisms. METHODS AND RESULTS: Whole-blood samples from 53 patients with GS were genotyped using PAP-PCR for UGT1A1 variations, including UGT1A160 (c.-3279T > G), UGT1A128 ([TA]6 > [TA]7), UGT1A16 (c.211G > A, G71R), UGT1A127 (c.686C > A, P229Q), UGT1A163 (c.1091C > T, P364L), and UGT1A17 (c.1456T > G, Y486D). For each locus, all cases were verified using the direct sequencing to assess PAP-PCR precision. One mutation type per locus was selected to investigate reproducibility and detection limits. The UGT1A1 gene polymorphism detected using PAP-PCR showed that, among the 53 patients, 83.02% presented missense mutations at UGT1A160 (c.-3279T > G), 54.72% had heterozygous or homozygous insertions in the TATA box in the promoter, 52.38% had the UGT1A16 variant (c.211G > A, G71R). The results obtained using the PAP-PCR and direct sequencing methods were almost consistent among all patients with UGT1A1 gene variants. However, there were different results in the promoter region variants among 2 patients in which PAP-PCR showed [TA]7 homozygosity, whereas direct sequencing revealed [TA]7/[TA]6 heterozygosity. This inconsistent result had been confirmed to be caused by differences in DNA polymerase activity. CONCLUSIONS: PAP-PCR could effectively detect UGT1A1 gene polymorphisms associated with Gilbert's syndrome. And it had exhibited superior accuracy in the analysis of TA repeat sequences compared to direct sequencing method. Additionally, due to its reduced program complexity, easy execution, and simple standardization, PAP-PCR may be a highly favorable option for clinical diagnosis.